Physiologically based pharmacokinetic modeling of imatinib and N-desmethyl imatinib for drug-drug interaction predictions.

The first-generation tyrosine kinase inhibitor imatinib has revolutionized the development of targeted cancer therapy and remains among the frontline treatments, for example, against chronic myeloid leukemia. As a substrate of cytochrome P450 (CYP) 2C8, CYP3A4, and various transporters, imatinib is highly susceptible to drug-drug interactions (DDIs) when co-administered with corresponding perpetrator drugs. Additionally, imatinib and its main metabolite N-desmethyl imatinib (NDMI) act as inhibitors of CYP2C8, CYP2D6, and CYP3A4 affecting their own metabolism as well as the exposure of co-medications. This work presents the development of a parent-metabolite whole-body physiologically based pharmacokinetic (PBPK) model for imatinib and NDMI used for the investigation and prediction of different DDI scenarios centered around imatinib as both a victim and perpetrator drug. Model development was performed in PK-Sim® using a total of 60 plasma concentration-time profiles of imatinib and NDMI in healthy subjects and cancer patients. Metabolism of both compounds was integrated via CYP2C8 and CYP3A4, with imatinib additionally transported via P-glycoprotein. The subsequently developed DDI network demonstrated good predictive performance. DDIs involving imatinib and NDMI were simulated with perpetrator drugs rifampicin, ketoconazole, and gemfibrozil as well as victim drugs simvastatin and metoprolol. Overall, 12/12 predicted DDI area under the curve determined between first and last plasma concentration measurements (AUClast) ratios and 12/12 predicted DDI maximum plasma concentration (Cmax) ratios were within twofold of the respective observed ratios. Potential applications of the final model include model-informed drug development or the support of model-informed precision dosing.

Application of a physiologically based pharmacokinetic model of rivaroxaban to prospective simulations of drug-drug-disease interactions with protein kinase inhibitors in cancer-associated venous thromboembolism.

AIMS: Rivaroxaban is a viable anticoagulant for the management of cancer-associated venous thromboembolism (CA-VTE). A previously verified physiologically-based pharmacokinetic (PBPK) model of rivaroxaban established how its multiple pathways of elimination via both CYP3A4/2J2-mediated hepatic metabolism and organic anion transporter 3 (OAT3)/P-glycoprotein-mediated renal secretion predisposes rivaroxaban to drug-drug-disease interactions (DDDIs) with clinically relevant protein kinase inhibitors (PKIs). We proposed the application of PBPK modelling to prospectively interrogate clinically significant DDIs between rivaroxaban and PKIs (erlotinib and nilotinib) for dose adjustments in CA-VTE. METHODS: The inhibitory potencies of the PKIs on CYP3A4/2J2-mediated metabolism of rivaroxaban were characterized. Using prototypical OAT3 inhibitor ketoconazole, in vitro OAT3 inhibition assays were optimized to ascertain the in vivo relevance of derived transport inhibitory constants (Ki ). Untested DDDIs between rivaroxaban and erlotinib or nilotinib were simulated. RESULTS: Mechanism-based inactivation (MBI) of CYP3A4-mediated rivaroxaban metabolism by both PKIs and MBI of CYP2J2 by erlotinib were established. The importance of substrate specificity and nonspecific binding to derive OAT3-inhibitory Ki values of ketoconazole and nilotinib for the accurate prediction of interactions was illustrated. When simulated rivaroxaban exposure variations with concomitant erlotinib and nilotinib therapy were evaluated using published dose-exposure equivalence metrics and bleeding risk analyses, dose reductions from 20 to 15 and 10 mg in normal and mild renal dysfunction, respectively, were warranted. CONCLUSION: We established a PBPK-DDDI model to prospectively evaluate clinically relevant interactions between rivaroxaban and PKIs for the safe and efficacious management of CA-VTE.

Toxicokinetic and bioavailability studies on retrorsine in mice, and ketoconazole-induced alteration in toxicokinetic properties.

Retrorsine (RTS) is a toxic retronecine-type pyrrolizidine alkaloid, which is widely distributed. The purpose of this study was to develop a high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum RTS determination in mice. Serum samples were deproteinated by acetonitrile, separated on a C18 -PFP column and delivered at 0.8 ml/min with an eluting system composed of water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid as mobile phases. RTS and the internal standard S-hexylglutathione (H-GSH) were quantitatively monitored with precursor-to-product transitions of m/z 352.1 → 120.1 and m/z 392.2 → 246.3, respectively. The method showed excellent linearity over the concentration range 0.05-50 µg/ml, with correlation coefficient r2 = 0.9992. The extraction recovery was >86.34%, and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <4.99%. The validated LC-MS/MS method was successfully applied to study the toxicokinetic profiles of serum RTS in mice after intravenous, oral administration and co-treated with ketoconazole, which showed that RTS displayed a long half-life (~11.05 h) and good bioavailability (81.80%). Co-administration of ketoconazole (KTZ) increased the peak serum concentration and area under the concentration-time curve and decreased the clearance and mean residence time. Summing up, a new standardized method was established for quantitative determination of RTS in sera.

Differential Impacts of Azole Antifungal Drugs on the Pharmacokinetic Profiles of Dasatinib in Rats by LC-MS-MS.

BACKGROUND: Dasatinib, as an oral multi-targeted inhibitor of BCR-ABL and SRC family kinases, has been widely used for the treatment of Philadelphia Chromosome Positive Leukemias in imatinib-acquired resistance and intolerance. The study aimed to develop and validate a simple and robust assay with a small volume of plasma based on liquid chromatography coupled with tandem mass spectrometry to determine the concentration of dasatinib and to investigate the impact of the cytochrome 3A4 inhibitors, including ketoconazole, voriconazole, itraconazole and posaconazole, on the pharmacokinetics of dasatinib in rats. METHODS: Thirty rats were divided randomly into five groups, control group (0.5% carboxymethylcellulose sodium), ketoconazole (30 mg/kg) group, voriconazole group (30 mg/kg), itraconazole group (30 mg/kg) and posaconazole group (30 mg/kg). After 150 µL blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 h and precipitated with acetonitrile, the plasma concentration of dasatinib was determined through Fluoro- Phenyl column (150 mm×2.1 mm, 3 µm) in a positive ionization mode. RESULTS: The results suggested that ketoconazole, voriconazole, and posaconazole could increase the AUC0-t of dasatinib to varying degrees while significantly reducing its clearance. However, there was no significant impact on the pharmacokinetics of dasatinib, co-administered with itraconazole except for the CL and MRT0-t of dasatinib. Additionally, voriconazole could significantly increase Cmax of dasatinib by approximately 4.12 fold. CONCLUSION: These data indicated that ketoconazole, posaconazole and voriconazole should be cautiously co-administered with dasatinib or close therapeutic drug monitoring of dasatinib concentration, which might cause the drug-drug interaction.

Physiologically based pharmacokinetic modeling and simulation to predict drug-drug interactions of ivosidenib with CYP3A perpetrators in patients with acute myeloid leukemia.

PURPOSE: Develop a physiologically based pharmacokinetic (PBPK) model of ivosidenib using in vitro and clinical PK data from healthy participants (HPs), refine it with clinical data on ivosidenib co-administered with itraconazole, and develop a model for patients with acute myeloid leukemia (AML) and apply it to predict ivosidenib drug-drug interactions (DDI). METHODS: An HP PBPK model was developed in Simcyp Population-Based Simulator (version 15.1), with the CYP3A4 component refined based on a clinical DDI study. A separate model accounting for the reduced apparent oral clearance in patients with AML was used to assess the DDI potential of ivosidenib as the victim of CYP3A perpetrators. RESULTS: For a single 250 mg ivosidenib dose, the HP model predicted geometric mean ratios of 2.14 (plasma area under concentration-time curve, to infinity [AUC0-∞]) and 1.04 (maximum plasma concentration [Cmax]) with the strong CYP3A4 inhibitor, itraconazole, within 1.26-fold of the observed values (2.69 and 1.0, respectively). The AML model reasonably predicted the observed ivosidenib concentration-time profiles across all dose levels in patients. Predicted ivosidenib geometric mean steady-state AUC0-∞ and Cmax ratios were 3.23 and 2.26 with ketoconazole, and 1.90 and 1.52 with fluconazole, respectively. Co-administration of the strong CYP3A4 inducer, rifampin, predicted a greater DDI effect on a single dose of ivosidenib than on multiple doses (AUC ratios 0.35 and 0.67, Cmax ratios 0.91 and 0.81, respectively). CONCLUSION: Potentially clinically relevant DDI effects with CYP3A4 inducers and moderate and strong inhibitors co-administered with ivosidenib were predicted. Considering the challenges of conducting clinical DDI studies in patients, this PBPK approach is valuable in ivosidenib DDI risk assessment and management.

Physiologically based pharmacokinetic modeling to assess metabolic drug-drug interaction risks and inform the drug label for fedratinib.

PURPOSE: Fedratinib (INREBIC®), a Janus kinase 2 inhibitor, is approved in the United States to treat patients with myelofibrosis. Fedratinib is not only a substrate of cytochrome P450 (CYP) enzymes, but also exhibits complex auto-inhibition, time-dependent inhibition, or mixed inhibition/induction of CYP enzymes including CYP3A. Therefore, a mechanistic modeling approach was used to characterize pharmacokinetic (PK) properties and assess drug-drug interaction (DDI) potentials for fedratinib under clinical scenarios. METHODS: The physiologically based pharmacokinetic (PBPK) model of fedratinib was constructed in Simcyp® (V17R1) by integrating available in vitro and in vivo information and was further parameterized and validated by using clinical PK data. RESULTS: The validated PBPK model was applied to predict DDIs between fedratinib and CYP modulators or substrates. The model simulations indicated that the fedratinib-as-victim DDI extent in terms of geometric mean area under curve (AUC) at steady state is about twofold or 1.2-fold when strong or moderate CYP3A4 inhibitors, respectively, are co-administered with repeated doses of fedratinib. In addition, the PBPK model successfully captured the perpetrator DDI effect of fedratinib on a sensitive CY3A4 substrate midazolam and predicted minor effects of fedratinib on CYP2C8/9 substrates. CONCLUSIONS: The PBPK-DDI model of fedratinib facilitated drug development by identifying DDI potential, optimizing clinical study designs, supporting waivers for clinical studies, and informing drug label claims. Fedratinib dose should be reduced to 200 mg QD when a strong CYP3A4 inhibitor is co-administered and then re-escalated to 400 mg in a stepwise manner as tolerated after the strong CYP3A4 inhibitor is discontinued.

Development and validation of a sensitive UHPLC-MS/MS analytical method for venetoclax in mouse plasma, and its application to pharmacokinetic studies.

A rapid and sensitive analytical method was developed to quantify venetoclax, an oral BH3-mimetic that blocks the anti-apoptotic protein BCL-2, in mouse plasma using ultra-high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection. Plasma protein precipitation was performed on 5 µL samples, and separation of the analytes was accomplished on an Accucore aQ column using gradient elution at a flow rate of 0.4 mL/min. The calibration curve was linear (R2 ≥ 0.99) over the concentration range of 5-1,000 ng/mL, and the lower limit of quantitation was 5 ng/mL. The intra-day and inter-day precisions (RSD%) were < 10.5%, and accuracies ranged from 94.4 to 106%. The developed method was successfully applied to pharmacokinetic studies involving serial 30 µL blood sampling from male and female mice after oral administration of venetoclax (10 mg/kg) alone or 30 min after oral administration of ketoconazole (50 mg/kg) or vehicle (PEG400). The observed pharmacokinetic profiles suggest venetoclax undergoes sexually dimorphic disposition in mice. However, regardless of sex, pharmacokinetic studies demonstrated that venetoclax AUC(0-6h) was increased greater than 2-fold with prior administration of ketoconazole. Overall, our pharmacokinetic studies suggest that mice could be a translationally relevant model for the characterization of venetoclax pharmacokinetics. We have developed an analytical method suitable for such murine pharmacokinetic studies.

Effects of repeated oral doses of ketoconazole on a sequential ascending single oral dose of fedratinib in healthy subjects.

PURPOSE: Fedratinib is an orally administered Janus kinase 2-selective inhibitor that is indicated for the treatment of adult patients with intermediate-2 or high-risk myelofibrosis in the United States. Fedratinib is metabolized by multiple cytochrome P450s (CYPs) in vitro, with the predominant contribution from CYP3A4. The primary objective of this study was to evaluate the effects of 14-day repeated 200 mg twice daily (BID) oral doses of a strong CYP3A4 inhibitor, ketoconazole, on a sequential ascending single oral dose of fedratinib in healthy male subjects. METHODS: An open-label, fixed-sequence, two-treatment cross-over study was conducted. Two cohorts of healthy adult males received two single doses of fedratinib (50 mg in Cohort 1 and 300 mg in Cohort 2) with one dose administered alone on Day 1 of Period 1 and the other dose coadministered with ketoconazole in the morning of Day 6 of Period 2. Subjects in both cohorts received 200-mg BID (Days 1-14) ketoconazole during Period 2. RESULTS: Coadministration of repeated 200-mg BID oral doses of ketoconazole for 14 days increased fedratinib exposure by 3.85- and 3.06-fold for area under the plasma concentration-time curve from time zero to infinity following a single oral dose of fedratinib of 50 and 300 mg, respectively. Oral administration of a single dose of 50 or 300 mg of fedratinib, administered alone or coadministered with steady-state ketoconazole, was safe and tolerable in the healthy male subjects. CONCLUSIONS: These results serve as the basis for fedratinib dose reduction when fedratinib is coadministered with strong CYP3A4 inhibitors.

Effects of CYP3A inhibitors on the pharmacokinetics of quizartinib, a potent and selective FLT3 inhibitor, and its active metabolite.

AIMS: Quizartinib is an oral, highly potent and selective next-generation FMS-like tyrosine kinase 3 (FLT3) inhibitor under investigation in patients with FLT3-internal tandem duplication-mutated acute myeloid leukaemia. This drug-drug interaction study assessed the pharmacokinetics (PK) of quizartinib when coadministered with strong or moderate cytochrome P450 3A (CYP3A) inhibitors. METHODS: In this parallel-group study, subjects were randomised to receive: (i) quizartinib + ketoconazole; (ii) quizartinib + fluconazole; or (iii) quizartinib alone. On Days 1-28, subjects received ketoconazole 200 mg or fluconazole 200 mg twice daily, and on Day 8, all subjects received a single 30-mg quizartinib dose. Blood samples were collected for PK analyses, steady-state PK parameters were simulated by superpositioning, and safety was assessed. RESULTS: Ninety-three healthy subjects were randomised; 86 completed the study. When administered with ketoconazole, geometric mean ratios (90% confidence interval) for quizartinib maximum observed plasma concentration (Cmax ) and area under the plasma concentration-time curve (AUC) from time 0 extrapolated to infinity were 117% (105%, 130%) and 194% (169%, 223%), respectively, vs quizartinib alone. Steady-state PK simulation demonstrated ~2-fold increase of both steady-state Cmax and AUC from time 0 to the end of the dosing interval when quizartinib was administered with ketoconazole due to accumulation of quizartinib at steady state. When administered with fluconazole, geometric mean ratios (90% confidence interval) for quizartinib Cmax and AUC from time 0 extrapolated to infinity were 111% (100%, 124%) and 120% (104%, 138%), respectively, vs quizartinib alone. Overall, 5.4% of subjects experienced quizartinib-related adverse events; no serious adverse events or deaths occurred. CONCLUSIONS: These results suggest reducing the dose of quizartinib when coadministered with a strong CYP3A inhibitor, but not with a moderate or weak CYP3A inhibitor. This dose reduction was implemented in phase 3 evaluation of quizartinib.

Osilodrostat Is a Potential Novel Steroidogenesis Inhibitor for the Treatment of Cushing Syndrome: An In Vitro Study.

CONTEXT: Metyrapone and ketoconazole, frequently used steroidogenesis inhibitors for treatment of Cushing syndrome, can be associated with side effects and limited efficacy. Osilodrostat is a CYP11B1 and CYP11B2 inhibitor, with unknown effects on other steroidogenic enzymes. OBJECTIVE: To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro. METHODS: HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells were incubated with osilodrostat, metyrapone, or ketoconazole (0.01 to 10 µM). Cortisol and ACTH were measured using chemiluminescence immunoassays, and steroid profiles by liquid chromatography-mass spectrometry. RESULTS: In HAC15 cells, osilodrostat inhibited cortisol production more potently (IC50: 0.035 µM) than metyrapone (0.068 µM; P < 0.0001), and ketoconazole (0.621 µM; P < 0.0001). IC50 values of osilodrostat and metyrapone for basal cortisol production varied with a 25- and 18-fold difference, respectively, with comparable potency. Aldosterone production was inhibited more potently by osilodrostat vs metyrapone and ketoconazole. Osilodrostat and metyrapone treatment resulted in strong inhibition of corticosterone and cortisol, 11-deoxycortisol accumulation, and modest effects on adrenal androgens. No pituitary-directed effects of osilodrostat were observed. CONCLUSIONS: Under our study conditions, osilodrostat is a potent cortisol production inhibitor in human adrenocortical cells, comparable with metyrapone. All steroidogenesis inhibitors showed large variability in sensitivity between primary adrenocortical cultures. Osilodrostat might inhibit CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 activity, and a proximal step in the steroidogenesis. Osilodrostat is a promising treatment option for Cushing syndrome, and in vivo differences with metyrapone are potentially driven by pharmacokinetic differences.

The effect of ketoconazole on praziquantel pharmacokinetics and the role of CYP3A4 in the formation of X-OH-praziquantel and not 4-OH-praziquantel.

AIM: The study sought to determine the effect of ketoconazole (KTZ) on the pharmacokinetics of praziquantel (PZQ) and on the formation of its major hydroxylated metabolites, cis- and trans-4-OH-PZQ, and X-OH-PZQ in healthy subjects. METHODS: Two treatments were evaluated by single-dose PK studies; the reference treatment was a 20 mg/kg dose of praziquantel given alone. The test treatment was a 20 mg/kg dose of praziquantel given in combination with 200 mg of ketoconazole. The study had a balanced and randomised cross-over design. Serial blood samples were collected between 0 and 12 h after each drug administration. PZQ, and cis- and trans-4-OH-PZQ and X-OH-PZQ concentrations in plasma were determined by LC-MS. A non-compartmental approach was used for pharmacokinetic analysis. Data were analysed using ANOVA and assessment of the 90% confidence interval of the geometric means of the log-transformed PK parameters obtained for each treatment. RESULTS: The pharmacokinetics of PZQ following the two treatments, PZQ alone and PZQ + KTZ, were not equivalent based on the assessment of the 90% CI of the geometric mean ratios of the AUC and Cmax (α = 0.05). The geometric mean ratios of the AUC and Cmax were found to be 176.8% and 227% respectively. The 90% CI of the AUC and Cmax were found to be 129.8%-239.8% and 151.4%-341.4% respectively. The AUC of PZQ was increased by 75% with KTZ co-administration (3516 vs 6172 ng h/ml) (p < 0.01). Meanwhile, the mean AUC of trans-4-OH-PZQ increased by 67% (61,749 ng h/ml vs 103,105 ng h/ml) (p < 0.01). X-OH-PZQ levels were reduced by about 57% (semi-quantified as 7311 ng h/ml vs 3109 ng h/ml by using trans-4-OH as standards) (p < 0.01) with KTZ co-administration. CONCLUSIONS: The relative bioavailability of praziquantel was increased by concomitant KTZ administration. KTZ preferentially inhibited the formation of X-OH-PZQ rather than 4-OH-PZQ, confirming in vitro data which implicates CYP3A4 in the formation of X-OH-PZQ rather than 4-OH-PZQ. The 4-hydroxylation of PZQ was shown to be the major metabolic pathway of PZQ, as evidenced by larger quantities of 4-OH-PZQ produced, thus explaining the modest albeit significant effect of ketoconazole on PZQ pharmacokinetics.

Formulation evaluation of ketoconazole microemulsion-loaded hydrogel with nigella oil as a penetration enhancer.

BACKGROUND: Onychomycosis is an opportunistic fungal infection often infecting people with compromised immune system. Currently available treatment interventions such as physical, surgical, and chemical-based approaches are successful in treating the condition, however, are painful and nonpatient complaint. Moreover, dermal creams with antifungal agents do not penetrate nail plate as required; hence, there is a necessity of developing a novel formulation with enhanced penetration. AIMS: The aim of the present research work was to develop ketoconazole microemulsion-loaded hydrogel formulation containing nigella oil as permeation enhancer for the treatment of onychomycosis. METHODS: Screening of oils, surfactants, and cosurfactants were done based on solubility studies followed by the construction of pseudo-ternary phase diagrams with 2% ketoconazole. The microemulsion was characterized for globule size, zeta potential, viscosity, and thermodynamic stability. Ex-vivo studies were carried out using Franz diffusion cells using porcine skin membrane. The antifungal activity of microemulsion-loaded hydrogel was evaluated using cup plate method using Candida albicans and Aspergillus niger. RESULTS: The optimized microemulsion had a composition of 54.97% Capryol:Nigella (2:1), 36.07% Transcutol:Propylene glycol (2:1), and 7.13% water and was later incorporated into polymeric gel base. The microemulsion-loaded hydrogel exhibited a 10 hours sustained release profile as compared to the marketed cream and an enhanced activity against marketed ketoconazole cream and compared with marketed ketoconazole formulation. CONCLUSION: The thermodynamic stability, sustained drug release with greater permeation, and enhanced activity due to the presence of nigella oil in microemulsion-loaded hydrogel warrant its application as an excellent vehicle for treating fungal infections.

Application of Physiologically Based Pharmacokinetic Modeling in Understanding Bosutinib Drug-Drug Interactions: Importance of Intestinal P-Glycoprotein.

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supraproportional at the lower doses (50-200 mg) and approximately dose-proportional at the higher doses (200-600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model on the basis of the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics on the basis of the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-glycoprotein inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute an element in the PBPK models critical to understanding the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates, such as bosutinib, that exhibit nonlinear pharmacokinetics owing largely to a saturation of intestinal P-glycoprotein-mediated efflux.

Simultaneous Physiologically Based Pharmacokinetic (PBPK) Modeling of Parent and Active Metabolites to Investigate Complex CYP3A4 Drug-Drug Interaction Potential: A Case Example of Midostaurin.

Midostaurin (PKC412) is being investigated for the treatment of acute myeloid leukemia (AML) and advanced systemic mastocytosis (advSM). It is extensively metabolized by CYP3A4 to form two major active metabolites, CGP52421 and CGP62221. In vitro and clinical drug-drug interaction (DDI) studies indicated that midostaurin and its metabolites are substrates, reversible and time-dependent inhibitors, and inducers of CYP3A4. A simultaneous pharmacokinetic model of parent and active metabolites was initially developed by incorporating data from in vitro, preclinical, and clinical pharmacokinetic studies in healthy volunteers and in patients with AML or advSM. The model reasonably predicted changes in midostaurin exposure after single-dose administration with ketoconazole (a 5.8-fold predicted versus 6.1-fold observed increase) and rifampicin (90% predicted versus 94% observed reduction) as well as changes in midazolam exposure (1.0 predicted versus 1.2 observed ratio) after daily dosing of midostaurin for 4 days. The qualified model was then applied to predict the DDI effect with other CYP3A4 inhibitors or inducers and the DDI potential with midazolam under steady-state conditions. The simulated midazolam area under the curve ratio of 0.54 and an accompanying observed 1.9-fold increase in the CYP3A4 activity of biomarker 4ß-hydroxycholesterol indicated a weak-to-moderate CYP3A4 induction by midostaurin and its metabolites at steady state in patients with advSM. In conclusion, a simultaneous parent-and-active-metabolite modeling approach allowed predictions under steady-state conditions that were not possible to achieve in healthy subjects. Furthermore, endogenous biomarker data enabled evaluation of the net effect of midostaurin and its metabolites on CYP3A4 activity at steady state and increased confidence in DDI predictions.

Assessment of effect of CYP3A inhibition, CYP induction, OATP1B inhibition, and high-fat meal on pharmacokinetics of the JAK1 inhibitor upadacitinib.

AIMS: Upadacitinib (ABT-494) is a selective Janus kinase 1 inhibitor being developed for treatment of auto-immune inflammatory disorders. This work evaluated effects of high-fat meal, cytochrome P450 (CYP) 3A inhibition, CYP induction, and organic anion transporting polypeptide (OATP) 1B inhibition on upadacitinib pharmacokinetics. METHODS: Two Phase 1 evaluations were conducted, each in 12 healthy subjects. In Study 1, using a randomized, two-sequence crossover design, a 3 mg dose of upadacitinib (immediate-release capsules) was administered alone under fasting conditions, after high-fat meal, or on Day 4 of a 6-day regimen of 400 mg once-daily ketoconazole. In Study 2, a 12 mg upadacitinib dose was administered alone, with the first, and with the eighth dose of a 9-day regimen of rifampin 600 mg once daily. Upadacitinib plasma concentrations were characterized. RESULTS: Administration of upadacitinib immediate-release capsules after a high-fat meal decreased upadacitinib Cmax by 23% and had no impact on upadacitinib AUC relative to the fasting conditions. Ketoconazole (strong CYP3A inhibitor) increased upadacitinib Cmax and AUC by 70% and 75%, respectively. Multiple doses of rifampin (broad CYP inducer) decreased upadacitinib Cmax and AUC by approximately 50% and 60%, respectively. A single dose of rifampin (also an OATP1B inhibitor) had no effect on upadacitinib AUC. Upadacitinib was well tolerated when co-administered with ketoconazole, rifampin, or after a high-fat meal. CONCLUSIONS: Strong CYP3A inhibition and broad CYP induction result in a weak and moderate effect, respectively, on upadacitinib exposures. OATP1B inhibition and administration of upadacitinib immediate-release formulation with food does not impact upadacitinib exposure.

Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions.

Drug interactions may dictate the failure or success of a treatment. Patients undergoing hematopoietic stem cell transplantation (HSCT) are exposed to various types of drugs, and understanding how these drugs interact is of the utmost importance. The pharmacokinetics of busulfan, melphalan, and cyclophosphamide, drugs commonly used for HSCT, are known to be affected by a variety of other drugs with differing molecular structures. We hypothesized that these structurally unrelated drugs affect the transport of DNA-alkylating agents. To test this hypothesis, we developed a flow cytometry assay that used 5-carboxyfluorescein diacetate acetoxymethyl ester, which is cleaved by nonspecific intracellular esterases to 5-carboxyfluorescein (5-CF), a fluorescent ligand for the drug transporter MRP1. A decreased 5-CF efflux in the presence of a test compound suggests competitive inhibition. We demonstrated that chlorambucil, 4-hydroperoxycyclophosphamide, ketoconazole, ethacrynic acid, everolimus, and sirolimus strongly inhibited 5-CF efflux in lymphoma and leukemia cell lines. The efflux of these drugs partially depends on the glutathione (GSH) level, and their cytotoxicity is synergistic with inhibited GSH synthesis. This is consistent with the hypothesis that their GSH-conjugated products are ligands of a common cellular drug transporter. Our results may explain clinical observations on the effects of various drugs on the pharmacokinetics and pharmacodynamics of alkylating agents, and the assay may be used to deduce interaction mechanisms of drugs transported by a common system.

Application of Physiologically Based Pharmacokinetic Modeling to the Understanding of Bosutinib Pharmacokinetics: Prediction of Drug-Drug and Drug-Disease Interactions.

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Philadelphia chromosome-positive chronic myelogenous leukemia. Bosutinib is predominantly metabolized by CYP3A4 as the primary clearance mechanism. The main objectives of this study were to 1) develop physiologically based pharmacokinetic (PBPK) models of bosutinib; 2) verify and refine the PBPK models based on clinical study results of bosutinib single-dose drug-drug interaction (DDI) with ketoconazole and rifampin, as well as single-dose drug-disease interaction (DDZI) in patients with renal and hepatic impairment; 3) apply the PBPK models to predict DDI outcomes in patients with weak and moderate CYP3A inhibitors; and 4) apply the PBPK models to predict DDZI outcomes in renally and hepatically impaired patients after multiple-dose administration. Results showed that the PBPK models adequately predicted bosutinib oral exposures in patients after single- and multiple-dose administrations. The PBPK models also reasonably predicted changes in bosutinib exposures in the single-dose DDI and DDZI results, suggesting that the PBPK models were sufficiently developed and verified based on the currently available data. Finally, the PBPK models predicted 2- to 4-fold increases in bosutinib exposures by moderate CYP3A inhibitors, as well as comparable increases in bosutinib exposures in renally and hepatically impaired patients between single- and multiple-dose administrations. Given the challenges in conducting numerous DDI and DDZI studies of anticancer drugs in patients, we believe that the PBPK models verified in our study would be valuable to reasonably predict bosutinib exposures under various scenarios that have not been tested clinically.

A Physiologically-Based Pharmacokinetic Modeling Approach To Predict Drug-Drug Interactions of Sonidegib (LDE225) with Perpetrators of CYP3A in Cancer Patients.

Sonidegib (Odomzo) is an orally available Smoothened inhibitor for the treatment of advanced basal cell carcinoma. Sonidegib was found to be metabolized primarily by cytochrome P450 (CYP)3A in vitro. The effect of multiple doses of the strong CYP3A perpetrators, ketoconazole (KTZ) and rifampin (RIF), on sonidegib pharmacokinetics (PK) after a single 800 mg dose in healthy subjects was therefore assessed. These data were used to verify a physiologically-based pharmacokinetic (PBPK) model developed to 1) bridge the clinical drug-drug interaction (DDI) study of sonidegib with KTZ and RIF in healthy subjects to the marketed dose (200 mg) in patients 2) predict acute (14 days) versus long-term dosing of the perpetrators with sonidegib at steady state and 3) predict the effect of moderate CYP3A perpetrators on sonidegib exposure in patients. Treatment of healthy subjects with KTZ resulted in an increased sonidegib exposure of 2.25- and 1.49-fold (area under the curve0-240h and maximal concentration respectively), and RIF decreased exposure by 72% and 54%, respectively. The model simulated the single- and/or multiple-dose PK of sonidegib (healthy subjects and patients) within ∼50% of observed values. The effect of KTZ and RIF on sonidegib in healthy subjects was also simulated well, and the predicted DDI in patients was slightly less and independent of sonidegib dose. At steady state, sonidegib was predicted to have a higher DDI magnitude with strong or moderate CYP3A perpetrators compared with a single dose. Different dosing regimens of sondigeb with the perpetrators were also simulated and provided guidance to the current dosing recommendations incorporated in the product label.

Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor.

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent. 2. Structure elucidation via high resolution, accurate mass LC-MS(n) revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction. 3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes. 4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.